Laser capture microdissection translation stage joystick

ABSTRACT

Systems and methods for laser capture microdissection are disclosed. An inverted microscope includes a translation stage joystick subsystem. The systems and methods provide the advantages of increased speed and much lower rates of contamination.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/121,677, filed Jul.23, 1998, now U.S. Pat. No. 6,639,657 which is a continuation of U.S.Ser. No. 09/018,452, filed Feb. 4, 1998, now U.S. Pat. No. 6,469,779,and claims benefit of both U.S. Ser. No. 60/060,731, filed Oct. 1, 1997and U.S. Ser. No. 60/037,864, filed Feb. 7, 1997, the entire contents ofall of which are hereby incorporated herein by reference as if fully setforth herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of laser capturemicrodissection (LCM). More particularly, the invention relates toinverted microscopes that include specialized apparatus for performingLCM. Specifically, a preferred implementation of the invention relatesto an inverted microscope that includes a cap handling subsystem, anillumination/laser optical subsystem, a vacuum chuck subsystem, and amanual joystick subsystem. The invention thus relates to invertedmicroscopes of the type that can be termed laser capture microdissectioninverted microscopes.

2. Discussion of the Related Art

Diseases such as cancer have long been identified by examining tissuebiopsies to identify unusual cells. The problem has been that there hasbeen no satisfactory prior-art method to extract the cells of interestfrom the surrounding tissue. Currently, investigators must attempt tomanually extract, or microdissect, cells of interest either byattempting to mechanically isolate them with a manual tool or through aconvoluted process of isolating and culturing the cells. Mostinvestigators consider both approaches to be tedious, time-consuming,and inefficient.

A new technique has been developed which can extract a small cluster ofcells from a tissue sample in a matter of seconds. The technique iscalled laser capture microdissection (LCM). Laser capturemicrodissection is a one-step technique which integrates a standardlaboratory microscope with a low-energy laser and a transparent ethylenevinyl acetate polymer thermoplastic film such as is used for the plasticseal in food product packaging.

In laser capture microdissection, the operator looks through amicroscope at a tissue biopsy section mounted on a standard glasshistopathology slide, which typically contains groups of different typesof cells. A thermoplastic film is placed over and in contact with thetissue biopsy section. Upon identifying a group of cells of interestwithin the tissue section, the operator centers them in a target area ofthe microscope field and then generates a pulse from a laser such as acarbon dioxide laser having an intensity of about 50 milliwatts (mW) anda pulse duration of between about 50 to about 500 milliseconds (mS). Thelaser pulse causes localized heating of the plastic film as it passesthrough it, imparting to it an adhesive property. The cells then stickto the localized adhesive area of the plastic tape directly above them,whereupon the cells are immediately extracted and ready for analysis.Because of the small diameter of the laser beam, extremely small cellclusters may be microdissected from a tissue section.

By taking only these target cells directly from the tissue sample,scientists can immediately analyze the gene and enzyme activity of thetarget cells using other research tools. Such procedures as polymerasechain reaction amplification of DNA and RNA, and enzyme recovery fromthe tissue sample have been demonstrated. No limitations have beenreported in the ability to amplify DNA or RNA from tumor cells extractedwith laser capture microdissection.

Laser capture microdissection has successfully extracted cells in alltissues in which it has been tested. These include kidney glomeruli, insitu breast carcinoma, a typical ductal hyperplasia of the breast,prostatic interepithielial neoplasia, and lymphoid follicles. The directaccess to cells provided by laser capture microdissection will likelylead to a revolution in the understanding of the molecular basis ofcancer and other diseases, helping to lay the groundwork for earlier andmore precise disease detection.

Another likely role for the technique is in recording the patterns ofgene expression in various cell types, an emerging issue in medicalresearch. For instance, the National Cancer Institute's Cancer GenomeAnatomy Project (CGAP) is attempting to define the patterns of geneexpression in normal, precancerous, and malignant cells. In projectssuch as CGAP, laser capture microdissection is a valuable tool forprocuring pure cell samples from tissue samples.

The LCM technique is generally described in the recently publishedarticle: Laser Capture Microdissection, Science, Volume 274, Number5289, Issue 8, pp 998–1001, published in 1996, the entire contents ofwhich are incorporated herein by reference. The purpose of the LCMtechnique is to provide a simple method for the procurement of selectedhuman cells from a heterogeneous population contained on a typicalhistopathology biopsy slide.

A typical tissue biopsy sample consists of a 5 to 10 micron slice oftissue that is placed on a glass microscope slide using techniques wellknown in the field of pathology. This tissue slice is a cross section ofthe body organ that is being studied. The tissue consists of a varietyof different types of cells. Often a pathologist desires to remove onlya small portion of the tissue for further analysis.

LCM employs a thermoplastic transfer film that is placed on top of thetissue sample. This film is manufactured containing organic dyes thatare chosen to selectively absorb in the near infrared region of thespectrum overlapping the emission region of common AlGaAs laser diodes.When the film is exposed to the focused laser beam the exposed region isheated by the laser and melts, adhering to the tissue in the region thatwas exposed. The film is then lifted from the tissue and the selectedportion of the tissue is removed with the film.

Thermoplastic transfer films such as a 100 micron thick ethyl vinylacetate (EVA) film available from Electroseal Corporation of PomptonLakes, N.J. (type E540) have been used in LCM applications. The film ischosen to have a low melting point of about 90° C.

The thermoplastic EVA films used in LCM techniques have been doped withdyes, such as an infrared napthalocyanine dye, available from AldrichChemical Company (dye number 43296-2 or 39317-7). These dyes have astrong absorption in the 800 nm region, a wavelength region thatoverlaps with laser emitters used to selectively melt the film. The dyeis mixed with the melted bulk plastic at an elevated temperature. Thedyed plastic is then manufactured into a film using standard filmmanufacturing techniques. The dye concentration in the plastic is about0.001 M.

While the films employed in LCM applications have proved satisfactoryfor the task, they have several drawbacks. The optical absorption of adye impregnated film is a function of its thickness. This property ofthe film may be in conflict with a desire to select film thickness forother reasons.

The organic dyes which are used to alter the absorption characteristicsof the films may have detrimental photochemistry effects in some cases.This could result in contamination of LCM samples. In addition, theorganic dyes employed to date are sensitive to the wavelength of theincident laser light and thus the film must be matched to the laseremployed.

BRIEF SUMMARY OF THE INVENTION

There is a particular need for an instrument that is well suited forlaser capture microdissection. There is also a particular need for animproved method of laser capture microdissection.

A first aspect of the invention is implemented in an embodiment that isbased on a laser capture microdissection method, comprising: providing asample that is to undergo laser capture microdissection; positioningsaid sample within an optical axis of a laser capture microdissectioninstrument; providing a transfer film carrier having a substrate surfaceand a laser capture microdissection transfer film coupled to saidsubstrate surface; placing said laser capture microdissection transferfilm in juxtaposition with said sample with a pressure sufficient toallow laser capture microdissection transfer of a portion of said sampleto said laser capture microdissection transfer film, without forcingnonspecific transfer of a remainder of said sample to said laser capturemicrodissection film; and then transferring a portion of said sample tosaid laser capture microdissection transfer film, without forcingnonspecific transfer of a remainder of said sample to said laser capturemicrodissection transfer film.

A second aspect of the invention is implemented in an embodiment that isbased on a laser capture microdissection instrument, comprising: aninverted microscope including: an illumination/laser optical subsystem;a translation stage coupled to said illuminator/laser optical subsystem;a cap handling subsystem coupled to said translation stage; a vacuumchuck subsystem coupled to said translation stage; and a manual joysticksubsystem coupled to said translation stage.

These, and other, aspects and objects of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingpreferred embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manychanges and modifications may be made within the scope of the inventionwithout departing from the spirit thereof, and the invention includesall such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

A clear conception of the advantages and features constituting theinvention, and of the components and operation of model systems providedwith the invention, will become more readily apparent by referring tothe exemplary, and therefore nonlimiting, embodiments illustrated in thedrawings accompanying and forming a part of this specification, whereinlike reference numerals (if they occur in more than one view) designatethe same elements. Consequently, the claims are to be given the broadestinterpretation that is consistent with the specification and thedrawings. It should be noted that the features illustrated in thedrawings are not necessarily drawn to scale.

FIG. 1 illustrates a perspective view of a laser capture microdissectioninverted microscope, representing an embodiment of the invention;

FIGS. 2A–2B illustrate orthographic views of the laser capturemicrodissection (LCM) inverted microscope shown in FIG. 1;

FIG. 3 illustrates a partial cross-sectional view of an LCM invertedmicroscope, representing an embodiment of the invention;

FIG. 4 illustrates a partial cross-sectional view of an LCM invertedmicroscope, representing an embodiment of the invention;

FIG. 5 illustrates a cross-sectional view of a cap handling subassembly,representing an embodiment of the invention;

FIG. 6 illustrates an elevational view of a cap handling subassembly ina load position, representing an embodiment of the invention;

FIG. 7 illustrates a top plan view of the apparatus in the positiondepicted in FIG. 6;

FIG. 8 illustrates an elevational view of a cap handling subassembly inan inspect position, representing an embodiment of the invention;

FIG. 9 illustrates a top plan view of the apparatus in the positiondepicted in FIG. 8;

FIG. 10 illustrates an elevational view of a cap handling subassembly inan unload position, representing an embodiment of the invention;

FIG. 11 illustrates a top plan view of the apparatus in the positiondepicted in FIG. 10;

FIG. 12 illustrates a top plan view of a vacuum chuck, representing anembodiment of the invention;

FIG. 13 illustrates a cross-sectional view of a vacuum chuck,representing an embodiment of the invention;

FIG. 14 illustrates a schematic diagram of a combined illuminationlight/laser beam delivery system, representing an embodiment of theinvention;

FIG. 15 illustrates a schematic view of a combined illumination/laserbeam delivery system with a diffuser in place, representing anembodiment of the invention;

FIG. 16 illustrates a schematic view of a combined illumination/laserbeam delivery system with a cap in place, representing an embodiment ofthe invention;

FIG. 17 illustrates a schematic view of an integrated cap/diffuser,representing an embodiment of the invention; and

FIG. 18 illustrates a schematic view of an integrated cap/diffuser,representing an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention and the various features and advantageous details thereofare explained more fully with reference to the nonlimiting embodimentsthat are illustrated in the accompanying drawings and detailed in thefollowing description. Descriptions of well-known components andprocessing techniques are omitted so as not to unnecessarily obscure theinvention in detail.

The entire contents of U.S. Ser. No. 60/037,864, filed Feb. 7, 1997entitled “Laser Capture Microdissection Device,” U.S. Ser. No.08/797,026, filed Feb. 7, 1997; U.S. Ser. No. 08/800,882, filed Feb. 14,1997; U.S. Ser. No. 60/060,731, filed Oct. 1, 1997; and U.S. Ser. No.60/060,732, filed Oct. 1, 1997 are hereby expressly incorporated byreference into the present application as if fully set forth herein.

Turning to FIG. 1, a perspective view of an inverted microscope 100 forlaser capture microdissection (LCM) is depicted. The inverted microscope100 includes a variety of subsystems, particularly adapted for the LCMtechnique, which combine to provide synergistic and unexpectedly goodresults. In alternative embodiments, the microscope does not need to bean inverted microscope.

A cap handling mechanic subassembly 110 provides structure for picking amicrocentrifuge tube cap 120 from a supply 122 and placing themicrocentrifuge tube cap 120 on top of a sample that is to undergo LCM.In the depicted embodiment, the microcentrifuge tube cap 120 is acylindrical symmetric plastic cap and the supply 122 includes eight ofthe consumables on a dovetail slide 124. In the depicted embodiment,there is a laser capture microdissection transfer film coupled to thebottom of the microcentrifuge tube cap 120. The cap handling mechanicsubassembly 110 is depicted in one of several possible positions whereina working end 112 of the cap handling mechanic subassembly 110 ispositioned in a vial capping station 114. The movement of the caphandling mechanic subassembly 110 will be described in more detailbelow.

A glass slide 130 upon which the sample to be microdissected is locatedand upon which the microcentrifuge tube cap 120 is placed, is located inthe primary optical axis of the inverted microscope 100. In alternativeembodiments, the slide that supports the sample can be made of othersubstantially transparent materials, for example, plastics such aspolycarbonate. The glass slide 130 is supported and held in place by avacuum chuck 140. The vacuum chuck 140 is a substantially flat surfacethat engages the glass slide 130 through a manifold (not shown) so as tohold the glass slide 130 in place while the microcentrifuge tube cap 120is picked and placed and while a translation stage 145 is manipulated inan X-Y plane. In alternative embodiments, the translation stage can beconfigured so as to have the capability of being moved along a Z axis.

The translation stage 145 can be manipulated using a pair of rotarycontrols (not shown in FIG. 1). In addition, the translation stage 145can be manipulated using a joystick 150. The joystick 150 is connectedto the translation stage 145 through a spherical mounting 152 and abracket 154. The joystick 150 includes a second spherical mounting 156within a static bracket 158. The joystick provides simultaneous X and Ymovement. Further, this simultaneous movement can be effected with in asingle-handed manner. The acquisition of samples is thus made quicker.

Mechanical leverage is provided by the fact that the length between thespherical mounting 152 and the second spherical mounting 156 is lessthan the length between the second spherical mounting 156 and the bottomend of the joystick 150. This leverage ratio is not needed formultiplication of force, but for the reduction in scalar movement. Thisratio should be less than 1/5, preferably approximately 1/7. This ratiocan be adjusted to provide the optimal resolution needed in terms ofsample movement as a function of operator hand movement.

In addition, the joystick provides tactile feedback not available withelectronic controls or geared linkages. The joystick 150 permitssimultaneous movement of the translation stage 145 in two directions (Xand Y) as a function of a single vector movement of an operator's hand.This important feature provides an unexpected result in that the speedwith which features to be microdissected can be positioned in theprincipal optical axis of the inverted microscope 100 is significantlyincreased.

Still referring to FIG. 1, the inverted microscope 100 includes an LCMoptical train 160. The LCM optical train 160 is mounted on anillumination arm 165. A white light illuminator 170 is also mounted onthe illumination arm 165. White light from the illuminator 170 passesdownward toward the microcentrifuge tube cap 120 through a dichroicmirror 180 and a focusing lens 190. A laser diode 175 with collimatingoptics emits a beam 177 that is reflected by a beam steering mirror 185.After the beam 177 is reflected by the beam steering mirror 185 it isincident upon the dichroic mirror 180. The dichroic mirror 180 is adichroic that reflects the beam 170 downward through the focusing lens190 toward the microcentrifuge tube cap 120. Simultaneously, thedichroic mirror 180 allows white light from the illuminator 170 to passdirectly down through the focusing lens 190 toward the microcentrifugetube cap 120. Thus, the beam 177 and the white light illumination aresuperimposed. The focusing lens 190 also adjusts the beam spot size.

Turning now to FIGS. 2A–2B, two orthographic views of the apparatusdepicted in FIG. 1 are illustrated. A white light illumination path 210and a laser beam path 220 can be seen in both FIGS. 2A and 2B. It can beappreciated from FIG. 2A that both of the paths include delivery ofoptical information to an image acquisition system 230. Similarly, itcan be appreciated from FIG. 2B that the illumination beam path includesdelivery of optical information to a binocular set 240. In alternativeembodiments, the eyepiece assembly (i.e., ocular) can include amonocular.

Turning to FIG. 3, a block schematic diagram of an optical trainaccording to the invention is depicted. A laser beam path 310 begins ata film activation laser 320. The laser beam path 310 is then reflectedby a mirror 330. The laser beam path 310 is then reflected by a dichroicmirror 340. The laser beam path 310 is then focused by a lens 350. Thelens 350 can optionally be associated with structure for changing thebeam diameter such as, for example, a variable aperture. The laser beampath 310 then passes downward toward the microcentrifuge tube cap 120.The laser beam path 310 then passes through an objective lens 360 and isthen reflected. A cut-off filter 390 is installed in the ocular 370. Thecut-off filter 390 can reflect and/or absorb the energy from the laserbeam.

The position of the laser beam path 310 with respect to the portion ofthe sample that is to be acquired by the microcentrifuge tube cap 120can be seen by an operator via the image acquisition system 230 (notshown in FIG. 3), which can include a camera. In idle mode, the laserbeam path 310 provides a visible low amplitude signal that can bedetected via the acquisition system 230. In pulse mode, the laser beampath 310 delivers energy to the microcentrifuge tube cap 120 and theoptical characteristics of the cut-off filter 390 attenuate the laserbeam path 310 sufficiently so that substantially none of the energy fromthe laser beam exits through ocular 370.

Suitable laser pulse widths are from 0 to approximately 1 second,preferably from 0 to approximately 100 milliseconds, more preferablyapproximately 50 milliseconds. In a preferred embodiment the wavelengthof the laser is 810 nanometers. In a preferred embodiment the spot sizeof the laser at the EVA material located on microcentrifuge tube cap 120is variable from 0.1 to 100 microns, preferably from 1 to 60 microns,more preferably from 5 to 30 microns. These ranges are relativelypreferred when designing the optical subsystem. From the standpoint ofthe clinical operator, the widest spot size range is the most versatile.A lower end point in the spot size range on the order of 5 microns isuseful for transferring single cells.

Suitable lasers can be selected from a wide power range. For example, a100 watt laser can be used. On the other hand, a 50 mW laser can beused. The laser can be connected to the rest of the optical subsystemwith a fiber optical coupling. Smaller spot sizes are obtainable usingdiffraction limited laser diodes and/or single mode fiber optics. Singlemode fiber allows a diffraction limited beam.

While the laser diode can be run in a standard mode such as TEM₀₀, otherintensity profiles can be used for different types of applications.Further, the beam diameter could be changed with a stepped lens insteadof lens 350.

Changing the beam diameter permits the size of the portion of the samplethat is acquired to be adjusted. Given a tightly focused initialcondition, the beam size can be increased by defocusing. Given adefocused initial condition, the beam size can be decreased by focusing.The change in focus can be in fixed amounts. The change in focus can beobtained by means of indents on a movable lens mounting and/or by meansof optical glass steps. In any event, increasing/decreasing the opticalpath length is the effect that is needed to alter the focus of the beam,thereby altering the spot size. For example, inserting a stepped glassprism 380 into the beam so the beam strikes one step tread will changethe optical path length and alter the spot size.

Turning now to FIG. 4, a schematic block diagram of another embodimentof an instrument according to the invention is depicted. In thisembodiment, a light source 410 (e.g., fluorescence laser), emits aspecific wavelength or wavelength range. The specific wavelength orwavelength range of a beam 420 emitted by the light source 410 ischosen, or filtered, to excite a fluorescent system (e.g., chemicalmarkers and optical filtering to techniques that are known in theindustry) that is incorporated in or applied to the sample to bemicrodissected. The frequency of a beam 420 emitted by the fluorescencelaser 410 can be tuned. The sample includes at least one member selectedfrom the group consisting of chromophores and fluorescent dyes(synthetic or organic), and, the process of operating the instrumentincludes identifying at least a portion of said sample with light thatexcites the at least one member, before the step of transferring saidportion of said sample to said laser capture microdissection transferfilm.

Still referring to FIG. 4, the beam 420 is reflected by a mirror 430.The beam 420 is then reflected by the dichroic mirror 340. In this waythe beam 420 can be made coincident with both the laser beam path 310and the white light from illuminator 170. It should be noted that thebeam 420 and the laser beam path 310 are shown in a spaced-apartconfiguration for clarity only. The beam 420 and the laser beam path 310can be coaxial. Fluorescence emitted by the sample beneath themicrocentrifuge tube cap 120 then travels through the objective lens 360to be viewed by the operator through ocular 370.

Turning now to FIG. 5, a cross-sectional view of the cap handlingmechanic subassembly 110 is depicted. The cap handling mechanicsubassembly 110 includes a dampener 510. The dampener 510 is a structurefor damping vertical motion of the cap handling mechanic subassembly110. The dampener 510 is adapted to lower the microcentrifuge tube cap120 down towards the translation stage in a reproducible manner. Thedampener 510 can be an air dampener (e.g., pneumatic tube) or liquiddampener (e.g., hydraulic tube) or any other dynamic structure capableor retarding the vertical motion of the subassembly 110 so as not togenerate an impulse. As the microcentrifuge tube cap 120 contacts theslide on which the sample rests (not shown), the working end 112 of anarm 520 that is coupled to the dampener 510 continues downward at areproducible rate. Therefore, the top of the microcentrifuge tube cap120 rises relative to the bottom of a weight 530. It can be appreciatedthat the cap 120 contacts the slide, before the weight 530 contacts thecap 120. In this way, the microcentrifuge tube cap 120 undergoes aself-leveling step before it is contacted and pressed against the slideby weight 530. As the weight 530 contacts the microcentrifuge tube cap120 the working end 112 of arm 520 continues along its downward path.Therefore, the application of the weight 530 to microcentrifuge tube cap120 is also a self-leveling step. By controlling the mass of weight 530,the force per unit area between the bottom of the microcentrifuge tubecap 120 and the slide can be controlled. After the sample on the slidehas undergone LCM, the arm 520 can be raised. By raising the arm, theweight 530 is first picked off the microcentrifuge tube cap 120 and thenthe microcentrifuge tube cap 120 is picked up off of the slide. Thedampener within the mechanism acts as a dash pot to control the velocityof the pickup arm.

The position of the translation stage is independent relative to theposition of the cap handling mechanic subassembly 110. These relativepositions can be controlled by the pair of rotary controls 147. It is tobe noted that the pair of rotary controls 147 are depicted with theiraxes parallel to the axis of the microcentrifuge tube cap 120 in FIG. 5.However, the pair of rotary controls 147 can be configured in anyorientation through the use of mechanical linkages such as gears.

Turning now to FIG. 6, the cap handling mechanic subassembly 110 isdepicted in a load position. In the load position, the working end 112of the arm 520 is located directly over the dovetail slide 124. In thisposition, the working end 112 grasps a microcentrifuge tube cap 120.After grasping the microcentrifuge tube cap 120, the arm 520 is raised,thereby picking the microcentrifuge tube cap 120 up.

Turning now to FIG. 7, a top plan view of the cap handling mechanicsubassembly 110 in the load position can be seen. Before the arm 520 isswung into the load position, a fresh microcentrifuge tube cap islocated beneath the axis of the working end 112. After the arm 520 isswung clockwise toward the vacuum chuck 140, the caps on dovetail slide124 will be advanced so as to position a fresh microcentrifuge tube capin place for the next cycle.

Turning now to FIG. 8, the cap handling mechanic subassembly 110 isdepicted in an inspect position. When positioned in the inspectposition, the working end 112 of the arm 520 is located coincident withthe principal optical axis of the instrument. This is the position inwhich the arm 520 is lowered to permit first the self-leveling of themicrocentrifuge tube cap 120 and then the self-leveling of the weight530 on top of the microcentrifuge tube cap 120. After LCM, the arm 520is raised in this position to put the weight 530 off the microcentrifugetube cap 120 and then the microcentrifuge tube cap 120 off the slide(not shown).

The weight 530 is a free-floating weight so that when it is set on topof the cap, the cap and weight are free to settle. The free-floatingweight permits the even application of pressure. For example, a weightof 30 grams can be used in the case where the total surface area of thelaser capture microdissection transfer film is approximately 0.26 squareinches.

Referring now to FIG. 9, a top plan view of the cap handling mechanicsubassembly 110 in the inspect position is depicted. It can beappreciated from this view that the working end 112 of the arm 520 islocated above the glass slide 130.

Turning now to FIG. 10, the cap handling mechanic subassembly 110 isdepicted in an unload position. In the unload position, the working end112 of the arm 520 and the cap 120 (aka consumable) with the LCMattached tissue are all located above the vial capping station 114.After being positioned on axis with the vial capping station 114, themicrocentrifuge tube cap 120 is lowered directly down onto, and into, ananalysis container 1000. After the microcentrifuge tube cap 120 isinserted into the analysis container 1000, the working end 112 of thearm 520 is raised up. The working end 112 of the arm 520 is then rotatedin a clockwise direction until it is above a fresh consumable(corresponding to the position depicted in FIGS. 6–7).

Turning now to FIG. 11, a top plan view of the cap handling mechanicsubassembly 110 in the unload position is depicted. In this position,the arm 520 is positioned away from the vacuum chuck 140. The analysiscontainer 1000 (not visible in FIG. 11) is pushed upward so as to engagethe microcentrifuge tube cap 120 (not visible in FIG. 11). The resultantsealed analysis container 1000 is then allowed to free-fall back into asupporting bracket 1010 (see FIG. 10). The sealed analysis container1000 together with the microcentrifuge tube cap 120 can then be takenfrom the bracket 1010 either manually or automatically.

Turning now to FIG. 12, a top plan view of the vacuum chuck 140 isdepicted. A top surface 1210 of the vacuum chuck 140 includes a firstmanifold hole 1020 and a second manifold hole 1030. In alternativeembodiments, there can be any number of manifold holes. The vacuum chuck140 includes a beam path hole 1040. When the instrument is in operation,the glass slide (not shown), or other sample holder, is placed over thebeam path hole 1040 and the manifold holes 1020–1030. After the glassslide is placed in position, a vacuum is pulled through a manifoldconnected to the holes 1020–1030, thereby holding the glass slide inposition over the beam path hole 1040. Although a medium or even a highvacuum can be applied, a low vacuum is sufficient to hold the glassslide in place during the LCM process. A sufficient vacuum can even begenerated with an inexpensive aquarium pump run in reverse.

The holding force exerted on the glass slide 130 is a function of theapplied vacuum, the size and shape of the manifold holes 1020–1030 andthe spacing between the top surface of the translation stage and thebottom surface of the glass slide 130. The spacing between thetranslation stage and the glass slide 130 is a function of the flatnessof the surfaces and the elasticity of the corresponding structures.

The level of vacuum is adjusted so that the glass slide 130, or othersample carrier, can be translated with regard to the translation stage.This translation capability is present when the vacuum is off and whenthe vacuum is on.

There is some leakage around the perimeter of the glass slide 130, whichmodulates the force holding the glass slide 130 in place. Accordingly, amoderate force (e.g., 5 pounds) applied to the edge of the glass slideis sufficient to cause movement of the glass slide 130 with regard tothe translation stage when the vacuum is engaged.

Turning now to FIG. 13, a cross section of the vacuum chuck is depictedwith a glass slide 130 in place. The vacuum that holds the glass slide130 in place is pulled through conduit 1320. The conduit 1320 isconnected to a circular manifold 1310. The circular manifold 1310 iscoupled with the manifold holes 1020–1030.

It can be appreciated from FIG. 13 that there are no pins, or otherstructures that project above the top surface of the vacuum chuck 140.This permits the glass slide 130 to be moved in any direction parallelwith the top surface without constraint.

Turning now to FIG. 14, a very high numerical aperture illuminator 1400for an LCM device is depicted. The illuminator 1400 provides a largeworking distance. A fiber optic 1410 provides a source of white lightillumination. The diverging beam 1420 from the fiber optic 1410 can havea numerical aperture of approximately 0.4. A collimator lens 1430collimates the light from the fiber optic 1410. The collimator lens 1430can be an aspheric lens (e.g., a Melles Griot (01 LAG 025) aspheric-likelens). A collimated beam 1440 from the collimator lens 1430 then passesthough a beam splitter 1450. The beam splitter 1450 permits theinjection of a laser beam 1460. After reflection by the beam splitter1450, the laser beam 1460 is coaxial with the white light illumination.Both types of light then reach a condenser lens 1470. Condenser lens1470 can be a Melles Griot (01 LAG 010) or (01 LAG 010) or other similaraspheric-like lens. The condensed coaxial beams are then incident uponand pass through the microcentrifuge tube cap 120. The focusing beamthat results from the condenser lens 1470 can have a numerical apertureof approximately 0.8. This can be characterized as a focusing beam. Themicrocentrifuge tube cap 120 is located on top of a slide with cells tobe sampled (not shown).

Turning now to FIG. 15, another embodiment of the high numericalaperture illuminator is depicted. In this embodiment, a diffuser 1500 islocated beneath the condenser lens 1470 at above the glass slide 130that contains the cells to be sampled.

More generally, any suitable scattering media can be used to provide thefunctions of the diffuser 1500. Providing such a scattering media nearthe tissue to scatter the light results in dramatically improvedillumination of the sample and much better visualization. A scatteringmedia of this type eliminates the need for refractive index matching ofthe sample. Such a scattering media can allow visualization of the cellnucleus and other subcellular structures that would normally be obscuredby normal illumination techniques.

The scattering media can be a diffuser material. A diffuser materialthat is suitable for use as the scattering media is milk or opal glass,which is a very dense, fine diffuser material. For instance, a suitablemilk glass is available from Edmund Scientific as Part No. P43,717.Standard laser printer/photocopier paper can even be used as thescattering media. Other types of transparent scattering media can beused, such as, for example, frosted glass, a lenticular sheet, a volumediffuser, and/or a surface diffuser. In any event, the scattering mediashould be a material that aggressively scatters the illumination light.A single sheet of typical ground glass is generally inadequate and needsto be combined in multiple layers as a serial stack of three or foursheets of ground glass to diffuse the illumination light sufficiently.

Turning now to FIG. 16, after the diffuser 1500 is replaced withmicrocentrifuge tube cap 120, the desired cells can be located using theimage acquired during the step represented in FIG. 15. Then, the laserbeam 1460 can be introduced, reflected off the beam splitter 1450 anddirected into the microcentrifuge tube cap 120 so as to acquire thedesired sample.

The purpose of the illuminator design is to provide a very highnumerical aperture illuminator for an LCM device. Such an LCM devicerequires a large working distance. While an illuminator that uses a 40×objective with 0.8 numerical aperture may seem to give bettervisualization, this design has problems since the working distance forthe 40× objective is very small, (e.g., less than 1 millimeter). Thus itis critical for a design that uses a thick dome carrier to have anillumination design with a much longer working distance. A thick domecarrier is a sample carrier whose top and bottom are spaced apart morethan a small distance. This is important because the sample is adjacentthe bottom of the sample carrier and the objective cannot move closer tothe sample than the top of the sample carrier.

The focusing lens 190 can be replaced with a Melles Griot asphericcondenser lens such as a 01 LAG 010. Such a lens has a numericalaperture of about 0.75 and a working distance of about 25 millimeters.Such a lens is not corrected for chromatic aberrations like the 40×objective. Experiments done using a spherical lens as a condenser gavegood improvement in visualization. This spherical lens clearly did nothave the corrections for aberrations that are built into the 40×objective.

The laser beam can be focused through this condenser lens like thefocusing lens 190. This condenser lens has roughly one-half the focallength of the current lens so the laser beam will be focused down toroughly 15 microns.

In an alternative embodiment the design could use a compound lens likethe lens in a barcode scanner. Such a compound lens would have a centralregion for the laser and a surrounding region that would act as a highnumerical aperture with regard to the white light illumination.

Turning now to FIG. 17, in one embodiment the diffuser 1500 can belocated adjacent to the microcentrifuge tube cap 120. In this embodimentthe microcentrifuge tube cap 120 is located just above the glass slide130. Collimated light 1700 is incident upon the diffuser 1500. Thediffuser 1500 causes the collimated light to enter into and pass throughthe cap at an infinite variety of angles. In this way, shadows arereduced and the quality of the imagery is improved.

The diffuser 1500 can be a volumetric diffuser or a surface diffuser. Inthe case of a volumetric diffuser, the diffuser 1500 can be frostedglass, a speckle based holographic diffuser or even a piece of paper. Inthe case of a surface diffuser, the diffuser 1500 can be a lenticularsheet, a speckle based holographic surface diffuser or any othersuitable topological surface.

Turning now to FIG. 18, the diffuser 1500 in this embodiment is locatedadjacent to the bottom of the microcentrifuge tube cap 120. Thecollimated light 1700 passes through the microcentrifuge tube cap 120and is incident upon the diffuser 1500. As the previously collimatedlight emerges from the diffuser 1500 it is scattered into a wide rangeof angles. In this embodiment, the diffuser 1500 is spaced apart fromthe glass slide 130.

The scattering media (e.g., diffuser 1500) can be directly or indirectlyconnected to the transfer film carrier and/or the LCM transfer film.Alternatively, the scattering media can be formed on a surface of, orthe interior of, the transfer film carrier and/or the LCM transfer film.The scattering media can be fabricated so as to shape the LCM beamand/or the illumination beam. The scattering media needs to be within afew millimeters of the sample to be effective. A few millimeters meansless than one centimeter, preferably less than five millimeters.

The process of operating the instrument begins by visualizing the tissuefrom which the sample is to be acquired. The tissue is then moved tobring the portion that is to be acquired directly below the principalaxis of the instrument. A laser capture microdissection transfer film isthen set over the desired area. In a preferred embodiment the film isspaced to within a few microns of the top surface of the sample.Alternatively, the film can be placed in contact with the top of thesample with a pressure sufficient to allow transfer without forcingnonspecific transfer. Finally, the laser is pulsed to heat the film andremove the tissue. The film needs to be pulled off of the samplequickly. Though the velocity should be such that the sample isthixotropically sheared.

Practical Applications of the Invention

A practical application of the invention that has value within thetechnological arts is the collection of a large database of geneexpression patterns of both healthy and diseased tissue, at differentstages of diseases. This database will be used to more fully understandthat pathogenesis of cancer and infectious diseases. The invention willenable a scientist to identify gene patterns and incorporate thisinformation into effective diagnostics for disease. The invention willallow medical doctors to compare actual patient tissue samples witharchived data from patient samples at different disease stages, therebyallowing them to prescribe more effective stage therapies, eliminateunnecessary procedures, and reduce patient suffering. Other researchareas where the invention will find use are drug discovery,developmental biology, forensics, botany, and the study of infectiousdiseases such a drug-resistant tuberculosis. There are virtuallyinnumerable uses for the invention, all of which need not be detailedhere.

Advantages of the Invention

A laser capture microdissection instrument and/or method representing anembodiment of the invention can be cost effective and advantageous forat least the following reasons. The invention will replace currentmethods with better technology that allows for more accurate andreproducible results. The invention can be used to provide a low costinjection molded polymer disposable that integrates a laser capturemicrodissection transfer film into the interior surface of an analysiscontainer such as a microcentrifuge tube (e.g., an EPPENDORF™ tube).

All publications, patent applications, and issued patents mentioned inthis application are hereby incorporated herein by reference in theirentirety to the same extent as if each individual publication,application, or patent was specifically and individually indicated to beincorporated in its entirety by reference.

All the disclosed embodiments of the invention described herein can berealized and practiced without undue experimentation. Although the bestmode of carrying out the invention contemplated by the inventors isdisclosed above, practice of the invention is not limited thereto. Itwill be manifest that various additions, modifications andrearrangements of the features of the invention may be made withoutdeviating from the spirit and scope of the underlying inventive concept.Accordingly, it will be appreciated by those skilled in the art that theinvention may be practiced otherwise than as specifically describedherein.

For example, the individual components need not be formed in thedisclosed shapes, or assembled in the disclosed configuration, but couldbe provided in virtually any shape, and assembled in virtually anyconfiguration. Further, the individual components need not be fabricatedfrom the disclosed materials, but could be fabricated from virtually anysuitable materials. Further, although the LCM instrument disclosedherein is described as a physically separate module, it will be manifestthat the LCM instrument may be integrated into other apparatus withwhich it is associated. Furthermore, all the disclosed elements andfeatures of each disclosed embodiment can be combined with, orsubstituted for, the disclosed elements and features of every otherdisclosed embodiment except where such elements or features are mutuallyexclusive.

It will be manifest that various additions, modifications andrearrangements of the features of the invention may be made withoutdeviating from the spirit and scope of the underlying inventive concept.It is intended that the scope of the invention as defined by theappended claims and their equivalents cover all such additions,modifications, and rearrangements. The appended claims are not to beinterpreted as including means-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase“means-for.” Expedient embodiments of the invention are differentiatedby the appended subclaims.

1. A laser microdissection instrument, comprising: a microscope havingan optical path; a laser coupled to the microscope; the laser having abeam path; a translation stage for receiving a sample; the translationstage being coupled to the microscope; a joystick connected to thetranslation stage; the joystick being configured to reduce a scalarmovement defined by an operator and having a leverage ratio to controlsample movement as a function of operator hand movement.
 2. The lasermicrodissection instrument of claim 1 wherein the joystick furtherincludes a handle, a first spherical mounting, a first bracket, a secondspherical mounting, and a second bracket; the translation stage isconnected to the first spherical mounting via the first bracket; thefirst spherical mounting is connected to the handle via the secondspherical mounting in the second static bracket; and the handle includesa bottom end.
 3. The laser microdissection instrument of claim 2 whereinthe distance between the first spherical mounting and the secondspherical mounting is less than the distance between the secondspherical mounting and the bottom end of the handle.
 4. The lasermicrodissection instrument of claim 1 wherein moving said sample andsaid translation stage with said joystick includes simultaneous X and Ymovement.
 5. The laser microdissection instrument of claim 1 wherein theleverage ratio is less than 1/5.
 6. The laser microdissection instrumentof claim 1 wherein the leverage ratio is less than 1/7.
 7. The lasermicrodissection instrument of claim 1 wherein the joystick is a manualjoystick.